2-Amino-6-(Difluoromethyl)-5,5-Difluoro-6-Phenyl-3,4,5,6-Tetrahydropyridines as BACE1 Inhibitors

ABSTRACT

The present invention is directed to compounds of formula (I) which are inhibitors of the BACE 1  enzyme. Separate aspects of the invention are directed to pharmaceutical compositions comprising said compounds and uses of the compounds to treat disorders for which the reduction of Aβ deposits is beneficial such as Alzheimer&#39;s disease.

FIELD OF THE INVENTION

The present invention provides compounds which act as BACE1 inhibitors.Separate aspects of the invention are directed to pharmaceuticalcompositions comprising said compounds and uses of the compounds totreat neurodegenerative or cognitive disorders.

BACKGROUND ART

Dementia is a clinical syndrome characterized by deficits in multipleareas of cognition that cannot be explained by normal aging, anoticeable decline in function, and an absence of delirium. In addition,neuropsychiatric symptoms and focal neurological findings are usuallypresent. Dementia is further classified based on etiology. Alzheimer'sdisease (AD) is the most common cause of dementia, followed by mixed ADand vascular dementia, Lewy body dementia (DLB), and fronto-temporaldementia. β-Amyloid deposits and neurofibrillary tangles are consideredto be major pathologic characterizations associated with AD which ischaracterized by the loss of memory, cognition, reasoning, judgment, andorientation. Also affected, as the disease progresses, are motor,sensory and linguistic abilities until global impairment of multiplecognitive functions occurs. β-Amyloid deposits are predominantly anaggregate of Aβ peptide, which in turn is a product of the proteolysisof amyloid precursor protein (APP) as part of the β-amyloidogenicpathway. Aβ peptide results from the cleavage of APP at the C-terminalby one or more γ-secretases and at the N-terminal by β-secretase 1(BACE1) also known as aspartyl protease 2. BACE1 activity is correlateddirectly to the generation of Aβ peptide from APP.

Studies indicate that the inhibition of BACE1 impedes the production ofAβ peptide. Further, BACE1 co-localizes with its substrate APP in Golgiand endocytic compartments (Willem M, et al. Semin. Cell Dev. Biol,2009, 20, 175-182). Knock-out studies in mice have demonstrated theabsence of amyloid peptide formation while the animals are healthy andfertile (Ohno M, et al. Neurobiol. Dis., 2007, 26, 134-145). Geneticablation of BACE1 in APP-overexpressing mice has demonstrated absence ofplaque formation, and the reverse of cognitive deficits (Ohno M, et al.Neuron; 2004, 41, 27-33). BACE1 levels are elevated in the brains ofsporadic AD patients (Hampel and Shen, Scand. J. Clin. Lab. Invest.2009, 69, 8-12).

These convergent findings indicate that the inhibition of BACE1 may be atherapeutic target for the treatment of AD as well as disorders forwhich the reduction of Aβ deposits is beneficial.

AstraZeneca announced the discovery of AZD3839, a potent BACE1 inhibitorclinical candidate for the treatment of AD (Jeppsson, F., et al. J.Biol. Chem., 2012, 287, 41245-41257) in October 2012. The effort whichled to the discovery of AZD3839 was further described in Ginman, T., etal. J. Med. Chem., 2013, 56, 4181-4205. The Ginman publication describesthe issues which were overcome in connection with the discovery andidentification of AZD3839. These issues related to poor blood brainbarrier penetration and P-glycoprotein mediated efflux of the compoundsresulting in lack of brain exposure.

The Ginman manuscript hypothesized that the differences in brainexposure would largely be due to the core structures and StructureActivity Relationship data was provided wherein the in vitro propertieson the reported compounds were given into four tables according to coresub-types. In table 4, a series of amidine containing compounds aredescribed that were considered interesting from an activity perspective.However, the data suggests that the amidine containing core did notexhibit a favourable blood brain barrier permeability profile.

Researchers from Hoffmann-La Roche and Siena Biotech also reported thediscovery of amidine containing compounds (Woltering, T. J., et al.Bioorg. Med. Chem. Lett. 2013, 23, 4239-4243). These compounds(compounds 17 and 18 in the paper) were found not to have any in vivoeffect (lack of Aβ40 reduction in brain in wild type mice).

Contrary to the teachings of Ginman, et al. and Woltering, T. J., etal., the inventors have discovered a series of amidine compounds whichare brain penetrant. Accordingly, the present invention relates to novelcompounds having BACE1 inhibitory activity, to their preparation, totheir medical use and to medicaments comprising them.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide compounds thatinhibit BACE1. Accordingly, the present invention relates to compoundsof Formula I.

wherein Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,isoxazolyl, and where the Ar is optionally substituted with one or moresubstituent selected from halogen, CN, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkenyl, C₁-C₆ fluoroalkyl or C₋- C₆ alkoxy; and

-   R1 is hydrogen, halogen, C₁-C₃ fluoroalkyl or C₁-C₃ alkyl;-   or a pharmaceutically acceptable salt thereof.

In one embodiment the present invention provides compounds of Formula Ior a pharmaceutically acceptable salt thereof for use in therapy.

The present invention further provides a pharmaceutical compositioncomprising a compound of Formula I or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier.

In one embodiment the invention provides the use of a compound ofFormula I or a pharmaceutically acceptable salt thereof in themanufacture of a medicament for the treatment of neurodegenerative orcognitive disorder.

In one embodiment, the invention provides a compound of Formula I or apharmaceutically acceptable salt thereof for use in a method for thetreatment of a neurodegenerative or cognitive disorder.

In one embodiment the present invention provides a method of treating aneurodegenerative or cognitive disorder comprising administering atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof to a patient in need thereof.

Further embodiments of the invention are provided immediately below:

-   In one embodiment, the compound is of formula Ia

-   or a pharmaceutically acceptable salt thereof.-   In one embodiment, R¹ is F or H, particularly F.-   In one embodiment, Ar is optionally substituted with one or more F,    Cl, Br, CN, C₁-C₃ alkyl, C₁-C₃ fluoroalkyl or C₁-C₃ alkoxy.-   In one embodiment, Ar is optionally substituted phenyl.-   In one embodiment, Ar is optionally substituted pyridyl.-   In one embodiment, Ar is optionally substituted pyrimidyl.-   In one embodiment, Ar is optionally substituted pyrazinyl.-   In one embodiment, Ar is optionally substituted imidazolyl.-   In one embodiment, Ar is optionally substituted pyrazolyl.-   In one embodiment, Ar is optionally substituted thiazolyl.-   In one embodiment, Ar is optionally substituted oxazolyl.-   In one embodiment, Ar is optionally substituted isoxazolyl.-   In one embodiment, the compound is selected from the group    consisting of:-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-methylthiaz    ole-2-carboxamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrimidine-2-carboxamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide-   or a pharmaceutically acceptable salt thereof.

A separate embodiment is directed to a pharmaceutical compositioncomprising a compound from the above list and a pharmaceuticallyacceptable carrier.

Another embodiment is directed to a method of treating aneurodegenerative or cognitive disorder comprising administering atherapeutically effective amount of a compound from the above list.

Yet another embodiment is directed to a use of a compound from the abovelist for the manufacture of a medicament for treating aneurodegenerative or cognitive disorder.

One embodiment is a compound from the above list for use in therapy.

Yet another embodiment is directed to a compound form the above list foruse in the treatment of a neurodegenerative or cognitive disorder.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of the compounds ofFormula I are inhibitors of BACE1, and as such, are useful for thetreatment of related disorders. Certain aspects of the invention areexplained in greater detail below but this description is not intendedto be a detailed catalog of all the different ways in which theinvention may be implemented, or all the features that may be added tothe instant invention. Hence, the following specification is intended toillustrate some embodiments of the invention, and not to exhaustivelyspecify all permutations, combinations and variations thereof.

As used herein, the term “C₁-C₆ alkyl” refers to a straight chained orbranched saturated hydrocarbon having from one to six carbon atomsinclusive. Examples of C₁-C₆ alkyl include, but are not limited to,methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-2-propyl,2-methyl-1-propyl, n-pentyl and n-hexyl. Similarly, the term “C₁-C₃alkyl” refers to a straight chained or branched saturated hydrocarbonhaving from one to three carbon atoms inclusive. Examples of suchsubstituents include, but are not limited to, methyl, ethyl andn-propyl.

Likewise, the term “C₁-C₆ alkoxy” refers to a straight chained orbranched saturated alkoxy group having from one to six carbon atomsinclusive with the open valency on the oxygen. Examples of C₁-C₆ alkoxyinclude, but are not limited to, methoxy, ethoxy, n-butoxy, t-butoxy andn-hexyloxy. The “C₁-C₆ alkoxy” is optionally substituted with one ormore fluorine atoms.

As used herein, the term “C₁-C₆ fluoroalkyl” refers to a straightchained or branched saturated hydrocarbon having from one to six carbonatoms inclusive substituted with one or more fluorine atoms. Examples ofC₁-C₆ fluoroalkyl include, but are not limited to, trifluoromethyl,pentafluoroethyl, 1 fluoroethyl, monofluoromethyl, difluoromethyl,1,2-difluoroethyl and 3,4 difluorohexyl. Similarly, the term “C₁-C₃fluoroalkyl” refers to a straight chained or branched saturatedhydrocarbon having from one to three carbon atoms inclusive substitutedwith one or more fluorine atoms per carbon atom.

The term “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “C₂-C₆ alkenyl” refers to a branched or unbranched alkenylgroup having from two to six carbon atoms and one double bond, includingbut not limited to ethenyl, propenyl, and butenyl. The term “C₂-C₆alkynyl” shall mean a branched or unbranched alkynyl group having fromtwo to six carbon atoms and one triple bond, including but not limitedto ethynyl, propenyl and butynyl.

As used herein, the phrase “effective amount” when applied to a compoundof the invention, is intended to denote an amount sufficient to cause anintended biological effect. The phrase “therapeutically effectiveamount” when applied to a compound of the invention is intended todenote an amount of the compound that is sufficient to ameliorate,palliate, stabilize, reverse, slow or delay the progression of adisorder or disease state, or of a symptom of the disorder or disease.In an embodiment, the method of the present invention provides foradministration of combinations of compounds. In such instances, the“effective amount” is the amount of a compound of the present inventionin the combination sufficient to cause the intended biological effect.

The term “treatment” or “treating” as used herein means ameliorating orreversing the progress or severity of a disease or disorder, orameliorating or reversing one or more symptoms or side effects of suchdisease or disorder. “Treatment” or “treating”, as used herein, alsomeans to inhibit or block, as in retard, arrest, restrain, impede orobstruct, the progress of a system, condition or state of a disease ordisorder. For purposes of this invention, “treatment” or “treating”further means an approach for obtaining beneficial or desired clinicalresults, where “beneficial or desired clinical results” include, withoutlimitation, alleviation of a symptom, diminishment of the extent of adisorder or disease, stabilized (i.e., not worsening) disease ordisorder state, delay or slowing of a disease or disorder state,amelioration or palliation of a disease or disorder state, and remissionof a disease or disorder, whether partial or total.

The present invention is based on the discovery that compounds ofFormula I are inhibitors of BACE1, and as such, are useful for thetreatment of disorders which pathological characteristics compriseβ-amyloid deposits and neurofibrillary tangles, such asneurodegenerative or cognitive disorders.

The compounds of the present invention are, as discussed above, expectedto be useful in the treatment of Alzheimer's disease due to theireffects on β-amyloid deposits and neurofibrillary tangles. This includesfamilial Alzheimer's disease where patients carry mutations on specificgenes intimately involved in the production of Aβ peptide. It is,however, important to note that aggregates of Aβ peptide is not limitedto familial Alzheimer's disease but is similarly an importantpathophysiological characteristics of the more common sporadicAlzheimer's disease [Mol Cell Neurosci, 66, 3-11, 2015].

The compounds of the present invention are also believed to be useful inthe treatment of early-stage Alzheimer's disease, i.e. disease stageswhere the biological and structural changes have started but theclinical manifestations of the disease have not yet become evident orare not yet well developed. Early-stage Alzheimer's disease may, infact, start years before any clinical manifestation of the diseasebecomes manifest. Early-stage Alzheimer's disease includes prodromalAlzheimer's disease, preclinical Alzheimer's disease and mild cognitiveimpairment. Although mild cognitive impairment may be unrelated toAlzheimer's disease it is often a transitional stage to Alzheimer'sdisease or due to Alzheimer's disease. Preclinical and prodromalAlzheimer's disease are asymptomatic stages, and they are typicallydiagnosed by the presence of Alzheimer's disease related biomarkers. Inthis context the compounds of the present invention are believed to beuseful in slowing down the progression of early-stage Alzheimer'sdisease, such as mild cognitive impairment to Alzheimer's disease. Thecompounds of the present invention are also believed to be useful in thetreatment of memory loss, attention deficits and dementia associatedwith Alzheimer's disease.

Other diseases, in addition to the continuum of Alzheimer's disease, arecharacterized by β-amyloid deposits and neurofibrillary tangles. Thisincludes e.g. Trisomy 21 also known as Down's syndrome. Patientssuffering from Down's syndrome have an extra chromosome 21 whichchromosome contains the gene for the amyloid precursor protein (APP).The extra chromosome 21 leads to overexpression of APP, which leads toincreased levels of Aβ peptide, which eventually causes the markedlyincreased risk of developing Alzheimer's disease seen in Down's syndromepatients [Alzheimer's & Dementia, 11, 700-709, 201]. Cerebral amyloidangiopathy is also characterized by β-amyloid deposits andneurofibrillary tangles in blood vessels of the central nervous system[Pharmacol Reports, 67, 195-203, 2015] and is as such expected to betreatable with compounds of the present invention.

In one embodiment, the present invention provides a method of treating adisease selected from Alzheimer's disease (familial or sporadic),preclinical Alzheimer's disease, prodromal Alzheimer's disease, mildcognitive impairment, Down's syndrome and cerebral amyloid angiopathy,the method comprising the administration of a therapeutically effectiveamount of a compound of Formula I or a pharmaceutically acceptable saltthereof to a patient in need thereof.

The present invention further provides a method of inhibiting BACE1 in apatient comprising administering to a patient in need thereof atherapeutically effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof.

The present invention also provides a method of inhibiting β-secretasemediated cleavage of amyloid precursor protein comprising administeringto a patient in need of such treatment a therapeutically effectiveamount a compound of Formula I or a pharmaceutically acceptable saltthereof. In further embodiments, the present invention provides the useof a compound of Formula I or a pharmaceutically acceptable salt thereoffor the manufacture of a medicament for the treatment of diseaseselected from Alzheimer's disease (familial or sporadic), preclinicalAlzheimer's disease, prodromal Alzheimer's disease, mild cognitiveimpairment, Down's syndrome or cerebral amyloid angiopathy.

The present invention also provides the use of a compound of Formula Ior a pharmaceutically acceptable salt thereof for the manufacture of amedicament for the inhibition of BACE1. The present invention furtherprovides the use of a compound of Formula I or a pharmaceuticallyacceptable salt thereof for the manufacture of a medicament for theinhibition of production or accumulation of Aβ peptide.

In one embodiment, the present invention provides a compound of FormulaI or a pharmaceutically acceptable salt thereof for use in a method forthe treatment of a disease selected form Alzheimer's disease (familialor sporadic), preclinical Alzheimer's disease, prodromal Alzheimer'sdisease, mild cognitive impairment, Down's syndrome or cerebral amyloidangiopathy.

In one embodiment, the present invention relates to a compound ofFormula I or a pharmaceutically acceptable salt thereof for use in amethod for inhibiting of BACE1 or in a method for inhibiting ofproduction or accumulation of Aβ peptide.

In a further embodiment, the invention provides a pharmaceuticalformulation adapted for any of the above treatments and uses.

In one embodiment, a mammal is a human.

In one embodiment, the patient is a human patient.

The present invention also comprises salts of the present compounds,typically, pharmaceutically acceptable salts. Such salts includepharmaceutically acceptable acid addition salts. Acid addition saltsinclude salts of inorganic acids as well as organic acids.

Representative examples of suitable inorganic acids includehydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, sulfamic,nitric acids and the like. Representative examples of suitable organicacids include formic, acetic, trichloroacetic, trifluoroacetic,propionic, benzoic, cinnamic, citric, fumaric, glycolic, itaconic,lactic, methanesulfonic, maleic, malic, malonic, mandelic, oxalic,picric, pyruvic, salicylic, succinic, methane sulfonic, ethanesulfonic,tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic,gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic,p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids,theophylline acetic acids, as well as the 8-halotheophyllines (forexample, 8-bromotheophylline and the like). Further examples ofpharmaceutically acceptable inorganic or organic acid addition saltsinclude the pharmaceutically acceptable salts listed in S. M. Berge, etal., J. Pharm. Sci., 1977, 66, 2.

Furthermore, the compounds of this invention may exist in unsolvated aswell as in solvated forms with pharmaceutically acceptable solvents suchas water, ethanol and the like.

The compounds of the present invention may have one or more asymmetriccentres and it is intended that any optical isomers (i.e. enantiomers ordiastereomers), as separated, pure or partially purified optical isomersand any mixtures thereof including racemic mixtures, i.e. a mixture ofstereoisomeres, are included within the scope of the invention.

In this context is understood that when specifying the enantiomericform, then the compound is in enantiomeric excess, e.g. essentially in apure form. Accordingly, one embodiment of the invention relates to acompound of the invention having an enantiomeric excess of at least 60%,at least 70%, at least 80%, at least 85%, at least 90%, at least 96%,preferably at least 98%.

Racemic forms may be resolved into the optical antipodes by knownmethods, for example, by separation of diastereomeric salts thereof withan optically active acid, and liberating the optically active aminecompound by treatment with a base. Separation of such diastereomericsalts can be achieved, e.g. by fractional crystallization. The opticallyactive acids suitable for this purpose may include, but are not limitedto d- or 1-tartaric, mandelic or camphorsulfonic acids. Another methodfor resolving racemates into the optical antipodes is based uponchromatography on an optically active matrix. The compounds of thepresent invention may also be resolved by the formation andchromatographic separation of diastereomeric derivatives from chiralderivatizing reagents, such as, chiral alkylating or acylating reagents,followed by cleavage of the chiral auxiliary. Any of the above methodsmay be applied either to resolve the optical antipodes of the compoundsof the invention per se or to resolve the optical antipodes of syntheticintermediates, which can then be converted by methods described hereininto the optically resolved final products which are the compounds ofthe invention.

Additional methods for the resolution of optical isomers, known to thoseskilled in the art, may be used. Such methods include those discussed byJ. Jaques, A. Collet and S. Wilen in Enantiomers, Racemates, andResolutions, John Wiley and Sons, New York, 1981. Optically activecompounds can also be prepared from optically active starting materials.

The present invention further provides a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of Formula Ior a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier. The present invention also provides a pharmaceuticalcomposition comprising a therapeutically effective amount of one of thespecific compounds disclosed in the Experimental Section and apharmaceutically acceptable carrier.

The compounds of the invention may be administered alone or incombination with pharmaceutically acceptable carriers or excipients, ineither single or multiple doses. The pharmaceutical compositionsaccording to the invention may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 22^(th)Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2013.

Pharmaceutical compositions for oral administration include solid dosageforms such as capsules, tablets, dragees, pills, lozenges, powders andgranules. Where appropriate, the compositions may be prepared withcoatings such as enteric coatings or they may be formulated so as toprovide controlled release of the active ingredient such as sustained orprolonged release according to methods well known in the art. Liquiddosage forms for oral administration include solutions, emulsions,suspensions, syrups and elixirs. Pharmaceutical compositions forparenteral administration include sterile aqueous and nonaqueousinjectable solutions, dispersions, suspensions or emulsions as well assterile powders to be reconstituted in sterile injectable solutions ordispersions prior to use. Other suitable administration forms include,but are not limited to, suppositories, sprays, ointments, creams, gels,inhalants, dermal patches and implants.

Typical oral dosages range from about 0.01 to about 100 mg/kg bodyweight per day.

The compounds of this invention are generally utilized as the free baseor as a pharmaceutically acceptable salt thereof. A pharmaceuticallyacceptable salt of a compound of Formula I is prepared e.g. in aconventional manner by treating a solution or suspension of a free baseof Formula I with a molar equivalent of a pharmaceutically acceptableacid. Representative examples of suitable organic and inorganic acidsare described above.

Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solutions and various organic solvents.Examples of solid carriers include lactose, terra alba, sucrose,cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate,stearic acid and lower alkyl ethers of cellulose. Examples of liquidcarriers include, but are not limited to, syrup, peanut oil, olive oil,phospholipids, fatty acids, fatty acid amines, polyoxyethylene andwater. Similarly, the carrier or diluent may include any sustainedrelease material known in the art, such as glyceryl monostearate orglyceryl distearate, alone or mixed with a wax. The pharmaceuticalcompositions formed by combining the compounds of Formula I or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier are readily administered in a variety of dosage formssuitable for the disclosed routes of administration. The formulationsmay conveniently be presented in unit dosage form by methods known inthe art of pharmacy.

If a solid carrier is used for oral administration, the preparation maybe tabletted, placed in a hard gelatin capsule in powder or pellet formor it may be in the form of a troche or lozenge. The amount of solidcarrier will vary widely but will range from about 25 mg to about 1 gper dosage unit. If a liquid carrier is used, the preparation may be inthe form of a syrup, emulsion, soft gelatin capsule or sterileinjectable liquid such as an aqueous or non-aqueous liquid suspension orsolution.

EXPERIMENTAL SECTION

The compounds of the present invention of the general formula I, whereinR¹ and Ar are as defined above can be prepared by the methods outlinedin the following reaction schemes 1-4 and in the examples. In thedescribed methods, it is possible to make use of variants ormodifications, which are themselves known to chemists skilled in the artor could be apparent to the person of ordinary skill in this art.Furthermore, other methods for preparing compounds of the invention willbe readily apparent to the person skilled in the art in light of thefollowing reaction schemes and examples.

For example, Scheme 2 describe the use of selective protecting groupsduring the synthesis of the compounds of the invention. One skilled inthe art would be able to select the appropriate protecting group for aparticular reaction. Moreover, it may be necessary to incorporateprotection and deprotection strategies for substituents such as amino,amido, keto and hydroxyl groups in the synthetic methods described belowto synthesize the compounds of Formula I. Methods for protection anddeprotection of such groups are well known in the art, and may be foundin T. Green, et al., Protective Groups in Organic Synthesis, 1991,2^(nd) Edition, John Wiley & Sons, New York.

For compounds, which can exist as a mixture or equilibrium between twoor more tautomers, only one tautomer is represented in the schemes,although it may not be the most stable tautomer. For compounds, whichcan exist in enantiomeric, stereoisomeric or geometric isomeric formstheir geometric configuration is specified; otherwise the structurerepresents a mixture of stereoisomers.

Analytical LC-MS data was obtained using the following methods.

Method A:

LC-MS was run on Waters Aquity UPLC-MS consisting of Waters Aquityincluding column manager, binary solvent manager, sample organizer, PDAdetector (operating at 254 nM), ELS detector, and SQ-MS equipped withAPPI-source operating in positive ion mode.

LC-conditions: The column was Acquity UPLC BEH C18 1.7 μm; 2.1×150 mmoperating at 60° C. with 0.6 ml/minutes of a binary gradient consistingof water+0.05% trifluoroacetic acid (A) and acetonitrile+5% water+0.03%trifluoroacetic acid (B). Gradient: 0.00 min: 10% B; 3.00 min: 99.9% B;3.01 min: 10% B; 3.60 min: 10% B. Total run time: 3.60 min.

Method B:

LC-MS was run on Waters Acquity UPLC-MS consisting of Waters Acquityincluding column manager, binary solvent manager, sample organizer, PDAdetector (operating at 254 nm), ELS detector, and TQ-MS equipped withAPPI-source operating in positive ion mode. LC-conditions: The columnwas Acquity UPLC BEH C18 1.7 μm ; 2.1×50 mm operating at 60° C. with 1.2ml/minutes of a binary gradient consisting of water+0.05%trifluoroacetic acid (A) and acetonitrile+5% water+0.05% trifluoroaceticacid (B). Gradient: 0.00 min: 10% B; 1.00 min: 100% B; 1.01 min: 10% B;1.15 min: 10% B. Total run time: 1.15 min.

¹H NMR spectra were recorded at 600 MHz on a Bruker Avance AV-III-600instrument or at 400 MHz on a Bruker Avance AV-III-400 instrument or aVarian 400 instrument. Chemical shift values are expressed in ppm-valuesrelative. The following abbreviations are used for multiplicity of NMRsignals: s=singlet, d=doublet, t=triplet, q=quartet, dd=double doublet,ddd=double double doublet, dt=double triplet, br=broad, and m=multiplet.

As an example and wherein R¹ is fluorine in the ortho position of thephenyl ring, compounds of the general formula IV may be prepared asshown in Scheme 1.

where R¹ is as defined under formula I and R is an alkyl group such asmethyl or ethyl. Compounds of the general formula IV (Scheme 1) may beprepared by reacting compounds of the general formula II with ahalogen-metal exchange reagent such as butyllithium followed by additionto an ester of general formula III.

As an example and wherein R¹ is fluorine in the ortho position of thephenyl ring, compound of the general formula XVI may be prepared asshown in Scheme 2.

where R² and R³ are an alkyl group such as methyl or ethyl.

Compounds of the general formula VII (Scheme 2) may be prepared byreacting compounds of the general formula IV with a sulfinamide such asVI in the presence of a Lewis acid/drying agent such as titaniumtetraethoxide. Treatment of compounds of the general formula VII withcompounds of the general formula VIII such as ethyl bromodifluoroacetatein the presence of Zn powder or in the presence of diethyl zinc andtris(triphenylphosphine)rhodium(I) chloride gives compounds of thegeneral formula IX. Compounds of the general formula X are obtained fromcompounds of the general formula IX by treatment with a reducing agentsuch as diisobutylaluminium hydride. In some cases compound X might bein equilibrium with the hydrate form. Treatment of compounds of thegeneral formula X with conditions such as methyl2-(dimethoxyphosphoryl)-acetate in the presence of lithium chloride anda base such as N,N-diisopropylethylamine gives compounds of the generalformula XI. Compounds of the general formula XII are obtained byhydrogenation of compounds of the general formula XI in the precense ofa catalyst such as palladium on carbon. Compounds of the general formulaXIII are obtained by treatment of compounds of the general formula XIIwith an acid such as hydrochloric acid in methanol followed by treatmentwith potassium carbonate in methanol or heating in a solvent such astoluene. Compounds of the general formula XIII can be nitrated usingnitric acid to give compounds of the general formula XIV. Treatment ofcompounds of the general formula XIV with a reagent such as Lawesson'sreagent(2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide)gives compounds of the general formula XV. Reduction of the nitro groupof compounds of the general formula XV gives compounds of the generalformula XVI.

Compounds of the general formula XIV may also be prepared as shown inScheme 3. Starting from nitro substituted acetophenones of generalformula IVb, compounds of the general formula XIb may be prepared asdescribed in Scheme 2 Compounds of the general formula XIIb are obtainedby hydrogenation of compounds of the general formula XIb in the presenceof a catalyst such as palladium on carbon. Compounds of the generalformula XIV may be prepared as described Scheme 2 for the preparation ofcompounds of the general formula XIII from compounds of the generalformula XII. Protection of the aniline moiety of compounds of thegeneral formula XIV gives compounds of the general formula XIVb.Treatment of compounds of the general formula XIVb with a reagent suchas Lawesson's reagent(2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide)followed by deprotection of the aniline moiety gives compounds of thegeneral formula XVI.

where R¹ is as defined under formula I and R³ is an alkyl group such asmethyl or ethyl

Compounds of the general formula I may be prepared as shown in Scheme 4.

where R¹ and Ar are as defined under formula I.

Compounds of the general formula XIX may be prepared by reactingcompounds of the general formula XVI with a carboxylic acid chloride ofgeneral formula XVII or by reaction with a carboxylic acid of generalformula XVIII using procedures known to chemists skilled in the art.Treatment of compounds of the general formula XIX with ammonia givescompounds of the general formula I. In some cases, the addition of anoxidizing reagent such as tert-butyl hydroperoxide might be necessary tofacilitate the reaction.

PREPARATION OF INTERMEDIATES INTERMEDIATE:2,2-difluoro-1-(2-fluorophenyl)ethan-1-one

To a solution of 1-bromo-2-fluorobenzene (10.00 g, 57.14 mmol) in THF(200 mL) was added n-butyllithium (2.5 M, 24.00 mL) drop-wise at −78° C.over a period of 15 minutes under N₂. The mixture was stirred at −78° C.for 30 min. Ethyl 2,2-difluoroacetate (10.64 g, 85.71 mmol) was addeddropwise at −78° C. and stirred for 2 hours at −78° C. TLC showed nostarting material remained. Saturated aqueous NH₄Cl (15 mL) was addeddropwise at −78° C. The reaction mixture was warmed to 25° C., extractedwith ethyl acetate (100 mL, three times). The combined organic phaseswere washed with brine (30 mL), dried over anhydrous Na₂SO₄, filteredand concentrated in vacuo. The residue was purified by columnchromatography on silica gel (petroleum ether:ethyl acetate=95:5) toafford 2,2-difluoro-1-(2-fluorophenyl)ethan-1-one (5.60 g, 47.8% yield,85% purity). ¹H NMR (CDCl₃, 400 MHz): δ 7.99-7.95 (m, 1H), 7.70-7.64 (m,1H), 7.33 (t, J=7.6 Hz 2H), 7.24 (dd, J=10.8, 8.4 Hz, 1H), 6.59-6.32 (m,1H).

INTERMEDIATE:(R)—N-(2,2-difluoro-1-(2-fluorophenyl)ethylidene)-2-methylpropane-2-sulfinamide

To a solution of 2,2-difluoro-1-(2-fluorophenyeethan-1-one (5.60 g,32.16 mmol) and (R)-2-methyl-propane-2-sulfinamide (5.07 g, 41.81 mmol)in THF (110 mL), was added tetraethoxytitanium (14.67 g, 64.32 mmol) inone portion at 26° C. The yellow solution was stirred at 80° C. for 2.5hr. TLC (petroleum ether:ethyl acetate=3:1) showed no starting materialremained. The mixture was cooled to 26° C. Water (10 mL) was added tothe mixture and it was filtered and extracted with ethyl acetate (60 mL,three times). The organic layer was washed with brine, dried overNa₂SO₄, filtered and concentrated and then purified by columnchromatography on silica gel (petroleum ether:ethyl acetate=91:9) toafford(R)—N-2,2-difluoro-1-(2-fluorophenyl)ethylidene)-2-methylpropane-2-sulfinamide(5.60 g, 61.6% yield, 98.1% purity).

INTERMEDIATE: ethyl(S)-3-(((R)-tert-butylsulfinyeamino)-2,2,4,4-tetrafluoro-3-(2-fluoro-phenyebutanoate

To a solution of(R)—N-(2,2-difluoro-1-(2-fluorophenyeethylidene)-2-methylpropane-2-sulfinamide(4.60 g, 16.6 mmol), ethyl 2-bromo-2,2-difluoro-acetate (6.73 g, 33.18mmol) and Rh(PPh₃)₃Cl (469 mg, 498 μmol) in THF (90 mL) was added asolution of diethyl zinc (1 M in THF, 33 mL) dropwise at −78° C. over aperiod of 20 minutes under Ar, during which the temperature wasmaintained below −65° C. The reaction mixture was warmed to 0° C. over aperiod of 10 minutes and stirred at 0° C. for 2 hours. TLC (petroleumether/ethyl acetate=3:1) showed the starting material was consumedcompletely. The dark red solution was quenched by water (40 mL) and thenfiltered. The filtrate was extracted with ethyl acetate (100 mL, twice).The combined organic phases were washed with saturated brine (30 mL),dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Thecrude product was purified by column chromatography on silica gel(petroleum ether/ethyl acetate=4:1) to give ethyl(S)-3-(((R)-tert-butylsulfinyeamino)-2,2,4,4-tetrafluoro-3-(2-fluorophenyl)butanoate(4.26 g, 62.1% yield). ¹H NMR (DMSO-d6, 400 MHz): δ 7.65-7.31 (m, 1H),7.49-7.44 (m, 1H), 7.23-7.12 (m, 2H), 6.93-6.66 (m, 1H), 5.00 (s, 1H),4.39-4.29 (m, 2H), 1.39 (s, 9H), 1.32 (t, J=8.0 Hz, 3H).

INTERMEDIATE:(R)-2-methyl-N−((S)-1,1,3,3-tetrafluoro-2-(2-fluorophenyl)-4-oxobutan-2-yl)-propane-2-sulfinamide

To a solution of ethyl(S)-3-(((R)-tert-butylsulfinyl)amino)-2,2,4,4-tetrafluoro-3-(2-fluorophenyl)-butanoate(3.20 g, 7.97 mmol) in dry THF (35 mL) was added dropwise a solution ofDIBAL-H (diisobutylaluminium hydride) in toluene (1.0 M, 16 mL, 16 mmol)at −78° C. under N₂. The mixture was stirred at −78° C. for 2 hours. Thereaction was quenched carefully with methanol (3 mL) at −78° C. Thenwater (20 mL) and ethyl acetate (200 mL) were added and the mixture waswarmed to 25° C. The mixture was aged for 30 minutes. The resultingmixture was filtered through a Celite pad. The organic layer was washedwith brine and dried over Na₂SO₄. The organic layer was concentrated togive crude product(R)-2-methyl-N—((S)-1,1,3,3-tetrafluoro-2-(2-fluorophenyl)-4-oxobutan-2-yl)propane-2-sulfinamide,which was used immediately in the next step without furtherpurification.

INTERMEDIATE: ethyl(S)-5-(((R)-tert-butylsulfinyeamino)-4,4,6,6-tetrafluoro-5-(2-fluorophenyl)-hex-2-enoate

To a stirred suspension of LiCl (405 mg, 9.56 mmol) in acetonitrile (30mL) under N₂ were added ethyl 2-diethoxyphosphorylacetate (2.14 g, 9.56mmol) and DIPEA (N,N-diisopropylethylamine) (2.06 g, 15.94 mmol) at 0°C. After 20 min,(R)-2-methyl-N—((S)-1,1,3,3-tetrafluoro-2-(2-fluorophenyl)-4-oxo-butan-2-yl)propane-2-sulfinamide(2.85 g, 7.97 mmol) in acetonitrile (10 mL) was added dropwise to themixture at 0° C. and the mixture was stirred at 25° C. for 17.5 hours.The reaction mixture was concentrated to remove acetonitrile, water (50ml) was added and extracted with ethyl acetate (200 ml). The organiclayer was dried and evaporated. The crude product was purified by columnchromatography (petrolium ether:ethyl acetate=5:1 to 4:1) to affordethyl (S) -5-(((R)-tert-butylsulfiny/amino)-4,4,6,6-tetrafluoro-5-(2-fluorophenyl)hex-2-enoate(1.77 g, 46.8% yield).

INTERMEDIATE: ethyl(S)-5-(((R)-tert-butylsulfinyeamino)-4,4,6,6-tetrafluoro-5-(2-fluorophenyl)-hexanoate

To a solution of ethyl(S)-5-(((R)-tert-butylsulfinyl)amino)-4,4,6,6-tetrafluoro-5-(2-fluorophenyl)hex-2-enoate(1.77 g, 4.28 mmol) in ethyl acetate (100 mL) was added Pd/C (400 mg,10%). The black suspension was stirred at 25° C. for 18 hours under45-50 psi H₂. It was filtrated and concentrated to give ethyl(S)-5-(((R)-tert-butylsulfinyeamino)-4,4,6,6-tetrafluoro-5-(2-fluorophenyehexanoate(1.70 g, 95.5% yield) which was used in the next step immediatelywithout further purification.

INTERMEDIATE: (S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluorophenyl)piperidin-2-one

To a solution of ethyl(S)-5-(((R)-tert-butylsulfinyl)amino)-4,4,6,6-tetrafluoro-5-(2-fluorophenyl)-hexanoate(1.70 g, 4.09 mmol) in dichloromethane (15 mL) was added HCl/MeOH (4 M,17 mL). The colorless solution was stirred at 25° C. for 1 hour. TLCanalysis showed no starting material was left. The mixture wasconcentrated and the residue was dissolved in toluene. The resultingmixture was concentrated again to give 1.5 g of a colorless oil. Thisoil was dissolved in toluene (30 mL) and was stirred at 100° C. for 18hours. After the mixture was cooled to 25° C., it was concentrated togive the crude product which was purified by flash chromatography onsilica gel (petroleum ether:ethyl acetate=2:1) to give(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluorophenyl)piperidin-2-one(880 mg, 3.15 mmol, 73% yield).

INTERMEDIATE:(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidin-2-one

(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluorophenyl)piperidin-2-one(880 mg, 3.15 mmol) was suspended in trifluoroacetic acid (2.55 mL). Themixture was cooled to 0° C. and concentrated H₂SO₄ (2.46 g, 24.3 mmol)was added. Then, HNO₃ (661.61 mg, 6.30 mmol) was added dropwise. After 2hours of stirring at 25 ° C., the reaction mixture was poured onto 100 gice and basified to pH>11 using 5 M NaOH (aq). The suspension wasextracted with ethyl acetate (150 mL). The phases were separated and theaqueous layer was extracted with ethyl acetate (2×70 mL). The combinedorganic phases were washed with a solution of saturated aqueous NH₄Cl(30 mL) and water (30 mL), dried over MgSO₄, filtered, and concentratedunder reduced pressure to afford(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidin-2-one(1.00 g, crude).

INTERMEDIATE:(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidine-2-thione

To a solution of(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidin-2-one(1.00 g, 3.08 mmol) in toluene (5 mL) was added Lawesson's reagent(2,4-bis(4-methoxyphenyl)-1,3,2,4-dithia-diphosphetane-2,4-disulfide)(686 mg, 1.70 mmol). The mixture was stirred at 100° C. for 2 hours. TLCanalysis showed no starting material remained. The mixture wasconcentrated and the crude product was purified by flash chromatographyon silica gel (petroleum ether:ethyl acetate=5:1) to give(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidine-2-thione(1.00 g, 2.94 mmol, 95.4% yield).

INTERMEDIATE: (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thione

To a suspension of(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidine-2-thione(1.00 g, 2.94 mmol) in ethanol (15 mL) and water (4 mL) was added ironpowder (821 mg, 145 mmol) and NH₄Cl (786 mg, 14.7 mmol, 5.0 Eq). Theblack mixture was stirred at 60° C. for 18 hours. After the reactionmixture was cooled to 25 ° C., the crude product was filtered and theresidue was washed with ethanol (100 mL). The combined filtrates wereconcentrated and the resulting solid was dispersed in ethyl acetate (100mL). The mixture was filtered and the filtrate was washed with water (30mL), brine (20 mL) and concentrated. The crude product was purified byflash chromatography on silica (petroleum ether:ethyl acetate=3:1˜2:1)to give(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thione(819 mg, 2.51 mmol, 85.3% yield). ¹H NMR (DMSO-d6, 400 MHz): δ 10.97 (s,1H), 7.03-6.90 (m, 2H), 6.64-6.55 (m, 2H), 5.19 (s, 2H), 3.19-3.15 (m,1H), 3.03-2.94 (m, 1H), 2.35-2.24 (m, 2H).

INTERMEDIATE: methyl-d₃-5-(methoxy-d₃)pyrazine-2-carboxylate

Sodium (0.094 g, 4.10 mmol) was added in small portions methanol-d₄(2.94 ml) and the reaction mixture was stirred until all sodium hasreacted. The soultion was the added to another soultion ofmethyl-5-chloropyrazine-2-carboxylate (0.6 g, 3.48 mmol) in methanol-d₄(0.98 ml). The reaction mixture was stirred for 1.5 hours at roomtemperature. The reaction mixture was concentrated in vacuo. 2 ml ofwater was added. The mixture was extracted with ethyl acetate. Theorganic phase was washed with brine, dried over MgSO₄ and concentratedin vacuo to give methyl-d₃-5-(methoxy-d₃)pyrazine-2-carboxylate.

INTERMEDIATE: methyl 5-(methoxy-d₃)picolinate

Methyl 5-hydroxypicolinate (2.88 g, 18.8 mmol) was dissolved indimethylformamide (108 ml) under argon. Potassium carbonate (7.20 g,52.1 mmol) was added and the orange suspension was stirred for 45minutes at room temperature. Iodomethane-d₃ (1.41 ml, 22.6 mmol) wasadded. The reaction mixture was stirred for 2 hours. Water was added.The mixture was extracted with ethyl acetate. The organic phase waswashed with brine, dried over MgSO₄ and concentrated in vacuo andpurified by column chromatography on silica gel (heptane: ethyl acetate)to give methyl 5-(methoxy-d₃)picolinate.

PREPARATION OF THE COMPOUNDS OF THE INVENTION EXAMPLE 1(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide(Compound 1)

5-chloropicolinic acid (19 mg, 0.12 mmol) and HATU(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate) (67.4 mg, 0.177 mmol) was dissolved DMF (1mL). The reaction mixture was stirred for 5 minutes. Then(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thione(25mg, 0.081 mmol) and DIPEA (N,-di-isopropyl ethylamine) (52 mg, 0.07ml, 0.4 mmol) were added. The reaction mixture was stirred for 1 hour atroom temperature. Satureted aqueous NH₄C1 was added and the mixture wasextracted with ethyl acetate. The combined organic phases were washedwith brine, dried over MgSO₄, filtered, and concentrated under reducedpressure Ammonia in methanol (7M, 2mL) was added and the reactionmixture was stirred at 55° C. in a sealed vial overnight. The reactionmixture was concentrated under reduced pressure. The crude product waspurified by flash chromatography on silica (heptane:ethyl acetate). Theproduct was dissolved in ethyl acetate and washed 5 times with saturetedaqueous NaHCO3/water to remove thiourea byproducts. The combined organicphases were washed with brine, dried over MgSO₄, filtered, andconcentrated under reduced pressure to give(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide.¹H NMR (DMSO-d6, 600 MHz): δ 10.78 (s, 1H), 8.79 (dt, J=2.4, 1.1 Hz,1H), 8.20 (dd, J=8.4, 2.4 Hz, 1H), 8.16 (dd, J=8.4, 0.7 Hz, 1H),7.96-7.90 (m, 1H), 7.88 (dd, J=6.8, 2.7 Hz, 1H), 7.20 (dd, J=11.6, 8.8Hz, 1H), 6.74 (t, J=55.2 Hz, 1H), 6.38 (s, 2H), 2.51 (dt, J=3.7, 1.8 Hz,2H), 2.19-1.98 (m, 2H).

LC-MS (m/z) 433 (MH⁺); t_(R)=0.53 minutes (Method A)

EXAMPLE 2(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide(Compound 2)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-fluoropicolinic acid.

¹H NMR (600 MHz, DMSO) δ 10.72 (s, 1H), 8.74 (d, J=2.8 Hz, 1H), 8.24(dd, J=8.7, 4.6 Hz, 1H), 7.99 (td, J=8.7, 2.8 Hz, 1H), 7.95-7.91 (m,1H), 7.88 (dd, J=6.8, 2.7 Hz, 1H), 7.20 (dd, J=11.6, 8.8 Hz, 1H), 6.74(t, J=55.2 Hz, 1H), 6.38 (s, 2H), 2.56-2.50 (m, 2H), 2.22-1.98 (m, 2H).

LC-MS (m/z) 417.1 (MH⁺); t_(R)=0.49 minutes (Method A)

EXAMPLE 3 (S)—N-(3-(6-amino-2-(difluor omethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide(Compound 3)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-methoxypyrazine-2-carboxylic acid. ¹H NMR (600 MHz, DMSO) δ 10.61(s, 1H), 8.89 (d, J=1.3 Hz, 1H), 8.42 (d, J=1.3 Hz, 1H), 7.90 (m, 2H),7.20 (dd, J=11.6, 8.9 Hz, 1H), 6.86-6.62 (m, 1H), 6.37 (s, 2H), 4.02 (s,3H), 2.56-2.50 (m, 2H), 2.19-1.96 (m, 2H).

LC-MS (m/z) 430.1 (MH⁺); t_(R) =0.48 minutes (Method A)

EXAMPLE 4(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide(Compound 4)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 2-methyloxazole-4-carboxylic acid.

¹H NMR (600 MHz, DMSO) δ 10.25 (s, 1H), 8.64 (s, 1H), 7.82 (m, 2H), 7.16(dd, J=11.6, 8.7 Hz, 1H), 6.73 (t, J=55.2 Hz, 1H), 6.36 (s, 2H), 3.01(s, 1H), 2.59-2.41 (m, 5H), 2.19-1.96 (m, 2H).

LC-MS (m/z) 403 (MH⁺); t_(R)=0.42 minutes (Method A)

EXAMPLE 5(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide(Compound 5)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-methoxypicolinic acid.

¹H NMR (600 MHz, DMSO) δ 10.55 (s, 1H), 8.40 (d, J=2.9 Hz, 1H), 8.13 (d,J=8.7 Hz, 1H), 7.94-7.90 (m, 1H), 7.84 (dd, J=6.7, 2.7 Hz, 1H), 7.62(dd, J=8.7, 2.9 Hz, 1H), 7.18 (dd, J=11.6, 8.8 Hz, 1H), 6.85-6.61 (m,1H), 6.36 (s, 2H), 3.93 (s, 3H), 2.55-2.47 (m, 2H), 2.17-2.00 (m, 2H).

LC-MS (m/z) 429.1 (MH⁺); t_(R)=0.5 minutes (Method A)

EXAMPLE 6 (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-di fluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide(Compound 6)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-difluoromethyl)pyrazine-2-carboxylic acid.

¹H NMR (600 MHz, DMSO) δ 11.02 (s, 1H), 9.40 (d, J=1.3 Hz, 1H), 9.10 (s,1H), 7.96-7.91 (m, 2H), 7.38-7.17 (m, 2H), 6.75 (t, J=55.1 Hz, 1H), 6.38(s, 2H), 2.56-2.50 (m, 2H), 2.20-1.98 (m, 2H).

LC-MS (m/z) 450.1 (MH⁺); t_(R)=0.48 minutes (Method A)

EXAMPLE 7 (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide(Compound 7)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-cyanopicolinic acid.

¹H NMR (600 MHz, DMSO) δ 10.94 (s, 1H), 9.21 (d, J=1.3 Hz, 1H), 8.59(dd, J=8.2, 1.9 Hz, 1H), 8.29 (d, J=8.1 Hz, 1H), 7.96-7.88 (m, 2H), 7.22(dd, J=11.5, 8.8 Hz, 1H), 6.74 (t, J=55.0 Hz, 1H), 6.37 (s, 2H),2.57-2.48 (m, 2H), 2.07 (m, 2H).

LC-MS (m/z) 424.5 (MH⁺); t_(R)=0.45 minutes (Method B)

EXAMPLE 8(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-4-methylthiazole-2-carboxamide(Compound 8)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 4-methylthiazole-2-carboxylic acid.

¹H NMR (600 MHz, DMSO) δ 10.84 (s, 1H), 7.90 (dd, J=6.7, 2.5 Hz, 1H),7.88-7.83 (m, 1H), 7.70 (s, 1H), 7.19 (dd, J=11.5, 8.9 Hz, 1H), 6.74 (t,J=55.1 Hz, 1H), 6.38 (s, 2H), 2.59-2.46 (m, 5H), 2.18-1.95 (m, 2H).

LC-MS (m/z) 419.4 (MH⁺); t_(R)=0.47 minutes (Method B)

EXAMPLE 9 (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrimidine-2-carboxamide(Compound 9)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-methoxypyrimidine-2-carboxylic acid.

¹H NMR (600 MHz, DMSO) δ 10.73 (s, 1H), 8.73 (s, 2H), 7.95-7.91 (m, 1H),7.82 (dd, J=6.7, 2.5 Hz, 1H), 7.22-7.18 (m, 1H), 6.74 (t, J=55.2 Hz,1H), 6.37 (s, 2H), 4.03 (s, 3H), 2.58-2.50 (m, 2H), 2.18-1.99 (m, 2H).

LC-MS (m/z) 430.5 (MH⁺); t_(R)=0.39 minutes (Method B)

EXAMPLE 10(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide(Compound 10)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-methoxy-3-methylpyrazine-2-carboxylic acid.

¹H NMR (600 MHz, DMSO) δ 10.52 (s, 1H), 8.24 (d, J=0.6 Hz, 1H), 7.91(ddd, J=8.8, 4.1, 2.8 Hz, 1H), 7.74 (dd, J=6.8, 2.7 Hz, 1H), 7.18 (dd,J=11.7, 8.8 Hz, 1H), 6.85-6.62 (m, 1H), 6.38 (s, 2H), 3.99 (s, 3H), 2.76(d, J=0.5 Hz, 3H), 2.55-2.49 (m, 2H), 2.18-1.99 (m, 2H).

LC-MS (m/z) 444.5 (MH⁺); t_(R)=0.52 minutes (Method B)

EXAMPLE 11(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide(Compound 11)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-cyano-3-methylpicolinic acid.

¹H NMR (600 MHz, DMSO) δ 10.80 (s, J=27.6 Hz, 1H), 8.99 (d, J=1.2 Hz,1H), 8.40 (s, 1H), 7.99-7.87 (m, 1H), 7.82-7.69 (m, 1H), 7.36-7.13 (m,1H), 6.73 (t, J=55.0 Hz, 1H), 6.36 (s, 2H), 2.56-2.47 (m, 2H), 2.21-1.97(m, 2H).

LC-MS (m/z) 438.1 (MH⁺); t_(R) =0.49 minutes (Method B)

EXAMPLE 12(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide(Compound 12)

Prepared as in example 1 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-bromopicolinic acid.

¹H NMR (600 MHz, DMSO) δ 10.78 (s, 1H), 8.87 (dd, J=2.3, 0.7 Hz, 1H),8.33 (dd, J=8.4, 2.3 Hz, 1H), 8.09 (dd, J=8.4, 0.6 Hz, 1H), 7.94 (ddd,J=8.8, 4.1, 2.8 Hz, 1H), 7.89 (dd, J=6.8, 2.7 Hz, 1H), 7.21 (dd, J=11.6,8.8 Hz, 1H), 6.88-6.62 (m, 1H), 6.39 (s, 2H), 2.60-2.49 (m, 2H),2.19-1.96 (m, 2H).

LC-MS (m/z) 479.1 (MH⁺); t_(R)=0.53 minutes (Method B)

EXAMPLE 13(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide(Compound 13)

(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thione(25 mg, 0.081 mmol) in was dissolved in dichloromethane (1 mL) under anatmosphere of argon. Trimethylaluminum (52 μl, 0.105 mmol, 2 molar,toluene) was added slowly, then methyl-(methoxy-d3)picolinate (18 mg,0.11 mmol) in 0.5 mL dichloromethane was added. The reaction mixture wasstirred at room temperature for 2 hours. The reaction mixture was pouredinto cooled 4N HCl (aq). The mixture was extracted with ethyl acetate.The organic phase was washed with brine, dried over magnesium sulphateand concentrated in vacuo. 7M ammonia in methanol (4 mL) was added andthe reaction mixture was stirred in a sealed vial at 50° C. overnight.The reaction mixture was concentrated in vacuo and was purified by flashchromatography on silica gel (heptane/ethyl acetate) followd bypurification by preperative HPLC to obtain the title compound as thetrifluroacetic acid salt.

¹H NMR (600 MHz, DMSO) δ 11.13 (s, 1H), 10.75 (s, 1H), 9.98 (s, 1H),9.10 (s, 1H), 8.40 (dd, J=2.9, 0.5 Hz, 1H), 8.21 (ddd, J=8.9, 4.1, 2.6Hz, 1H), 8.15 (dd, J=8.7, 0.5 Hz, 1H), 8.09 (dd, J=6.8, 2.6 Hz, 1H),7.64 (dd, J=8.7, 2.9 Hz, 1H), 7.40 (dd, J=11.9, 9.0 Hz, 1H), 7.20 (t,J=53.0 Hz, 1H), 3.17-3.01 (m, 2H), 2.48-2.38 (m, 2H).

LC-MS (m/z) 432 (MH⁺); t_(R)=0.49 minutes (Method A)

EXAMPLE 14(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide(Compound 14)

Prepared as in example 13 from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand methyl 5-(methoxy-d₃)picolinate.

¹H NMR (600 MHz, DMSO) δ 10.61 (s, 1H), 8.89 (d, J=1.2Hz, 1H), 8.42 (d,J=1.2Hz, 1H), 7.91-7.86 (m, 2H), 7.19 (dd, J=11.5, 8.9 Hz, 1H),6.84-6.61 (m, 1H), 6.36 (s, 2H), 2.53-2.49 (m, 2H), 2.16-1.96 (m, 2H).

LC-MS (m/z) 433.1 (MH⁺); t_(R) =0.49 minutes (Method A)

Stereochemistry

Crystals were obtained by recrystallization of(S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidin-2-yl)-4-fluorophenyl)picolinamidefrom a mixture of heptane and ethyl acetate. The structure of(S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidin-2-yl)-4-fluorophenyl)picolinamide was elucidated by X-ray crystallography of said crystals.The structure shows the absolute and relative configuration of(S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidin-2-yl)-4-fluorophenyepic olinamide .(S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidin-2-yl)-4-fluorophenyepicolinamide was prepared as described in example 1 starting from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thioneand 5-bromopicolinic acid.

The absolute configurations of the exemplified compounds of the presentinvention can thus be rationalized.(S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidin-2-yl)-4-fluorophenyl)picolinamidewas prepared from(S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoropiperidine-2-thionewhich is staring material for all exemplified compounds of the presentinvention.

Pharmacological Testing

BACE1 Binding Assay

The binding assay was performed as SPA-based assay using a biotinylatedform of human BACE1 recombinantly expressed and subsequently purifiedfrom Freestyle HEK293 cells. The binding assay was run in a 50 mM sodiumacetate buffer, pH 4.5 containing 50 mM NaCl and 0.03% Tween-20 in whiteclear bottom 384 plates (Corning #3653). 10 nM (final concentration)radioligand([³H]-N-((1S,2R)-1-benzyl-3-cyclopropylamino-2-hydroxy-propyl)-5-(methanesulfonyl-methyl-amino)-N#R)-1-phenyl-ethyl)-isophthalamide)(TRQ11569 purchased from GE Healthcare) was mixed with test compound ata given concentration, 6 nM (final concentration) human BACE1 and 25 μgStreptavidin coated PVT core SPA beads (RPNQ0007, GE Healthcare LifeSciences) in a total volume of 40 μl. Several concentrations of eachtest compound were tested in the assay for IC₅₀ determination. Theplates were incubated for one hour at room temperature and counted in aWallac Trilux counter. Total and non-specific binding were determinedusing buffer and 1 μM (final concentration) of the high affinity BACE1reference inhibitor(S)-6-[3-chloro-5-(5-prop-1-ynyl-pyridin-3-yl)-thiophen-2-yl]-2-imino-3,6-dimethyl-tetrahydro-pyrimidin-4-one,respectively. For each test compound, a IC₅₀ value (the concentrationmediating 50% inhibition of the specific binding of the radioligand) wasdetermined from concentration-response curve and used to calculate theK_(i) from the equation K_(i)=IC₅₀/(1+L/K_(d)), where L and K_(d) arethe final concentration of the radioligand used in the assay and thedissociation constant of the radioligand, respectively. The K_(d) of theradioligand was determined from saturation binding experiments.

TABLE 1 binding affinity of selected compounds Compound BACE1 No Ki (nM)1 18 2 23 3 8.5 4 20 5 6.7 6 20 7 7.4 8 71 9 6.9 10 16 11 7.8 12 6.1 1314 14 18

BACE1 Efficacy Assay

The efficacy assay was performed as a FRET-based assay using acommercially available BACE1 kit (Life Technologies, P2985). 2 μl testcompound at 10 μM (final concentration) and 15 μl BACE1 enzyme from thekit (final concentration 3 nM) were preincubated for 15 minutes at roomtemperature before addition of 15 μl of substrate from the kit (250 nMfinal concentration) and incubated for additional 90 minutes at roomtemperature. The assay plate was subsequently read in a Pherastar(Ex540/Em590). The enzyme activity observed in presence of test compoundwere normalized to the enzyme activity observed in presence of bufferand 10 μM (final concentration) of the high affinity BACE1 referenceinhibitor (S)-6-[3 -Chloro-5-(5-prop-1-ynyl-pyridin-3-yl)-thiophen-2-yl]-2-imino-3,6-dimethyl-tetra-hydropyrimidin-4-one, respectively. Theefficacy of the test compounds was evaluated at 10 μM (finalconcentration) and defined as the percent inhibition of the enzymeactivity using the equation %inhibition=100% -normalized enzyme activityin percent.

TABLE 2 BACE1 activity of selected compounds BACE1 inhibition atCompoundNo 10 μM (%) 1 106 2 100 3 103 4 104 5 101 6 103 8 102 9 103 10106 13 103 14 108

Assessment of AD Levels in Rat Brain and Plasma Following BACE1Inhibition. Animals.

All rat care and experimental procedures were approved by LundbeckVeterinary Staff, according to Danish legislature. The rats weremaintained in a barrier facility with a 12/12-h light/dark cycle and adlibitum food and water access.

Treatment of Naïve Rats.

Young adult Male Sprague Dawley rats of approximately 250 g weight werepurchased from Charles River and received 0-30 mg/kg of vehicle (10% HPbetaCD+1M MeSO₄, pH 2.5) or test compounds (dissolved in vehicle) onlyby oral gavage (p.o). The compounds are dosed at a volume of 5 ml/kg.Cohorts of 5-10 animals were established for each treatment condition.

The animals undergoing treatment were closely monitored by veterinarystaff for any signs of toxicity. Monitoring parameters included bodyweight, physical appearance, changes in coat appearance, occurrence ofunprovoked behavior, and blunted or exaggerated responses to externalstimuli.

Tissue Collection.

At T=180 minutes after initial dosing the animals were stunned anddecapitated with a guillotine. Trunk-blood was sampled in EDTA coatedtubes after decapitation of the animal. The blood was centrifuged at2200G at 4° C. for 15 minutes and the plasma was collected and frozen at−80° C. The blood was aliquoted for Aβ ELISA and DMPK analysisImmediately following sacrifice, the brain was extracted and split into2 halves. The right hemibrains were snap frozen on dry ice and stored at−80° C. The left half was dissected; with the front forebrain taken forAβ ELISA and the remainder used for DMPK analysis. These samples werealso snap frozen on dry ice and stored at −80° C. until use foranalysis.

Tissue Processing.

The cortex samples were thawed slightly on wet ice before they werehomogenized with a small volume dispersing instrument (T10 basicULTRA-TURRAX®) which was set at speed 5 for approximately 5-7 sec. Thetissue was processed in a 10 times volume of the weight, for example 100mg of tissue was homogenized in 1000 μL of Homogenization buffer.Homogenization buffer: 50 ml Milli Q water+50 nM NaCl+0.2% Diethylamin(DEA)+1 tablet of Complete Protease inhibitor cocktail+1 nM4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride irreversibleserine protease inhibitor (AEBSF).

After homogenization 450 μL aliquots of the samples are collected into a1.5 ml Eppendorf tube and placed on wet ice, 0.5% NP-40 (50 ul ) wasadded to all samples and then they were incubated on ice for 30 min.After which all samples were sonicated using an Ultrasonic homogenizerwith 20 kHz homogeneous sound (SONOPLUS HD2070, Bandelin Electronic) 10pulse set at 12-13% power to extract all the Aβ species. The sampleswere then centrifuged (Ole Dich 157 MPRF Micro centrifuge) at 20000 Gfor 20 minutes at 4° C. After centrifugation 285 μL of the supernatantwas pipetted into 600 μL microtubes tubes and neutralized with 15 μL of1M Tris-HCL buffer.

ELISA Protocol.

WAKO 294-62501 Human/Rat Abeta amyloid (40) kit was used for all ELISAanalyses. 30 μL plasma samples or 30 μL of the cortex supernatantsgenerated as described above were placed in 600 μL microtubes tubes onwet ice. To this 30 μL of 8M Urea (AppliChem A1049, 9025) are added togenerate a 2-fold dilution. Both plasma and cortex supernatants areincubated on ice for 30 min. Standard rows were prepared from thestandard peptide stock provided in the kit and standard diluentcontaining 1.6M Urea (200 μL8M Urea+800 μL of standard diluent) and 0.8MUrea (400 μL 8M Urea+3600 μL Standard diluent). A serial 2-fold dilutionof Aβ40 from 100 pmol/ml to 0 pmol/L was prepared for the assay.

After incubation with urea, all samples were further diluted by additionof 5 times standard diluent from the Kit. This was done by adding 240 μLStandard Diluent to 60 μL sample/urea mixture, which was then mixedwell. 100 μL of each diluted sample was pipetted into designated wellsof the ELISA plate in duplicates. The plate was then covered andincubated overnight at 4° C. The following day, the ELISA kit wasbrought to room temperature before use. The incubated plate was washed 5times with the 20× washing solution diluted in Milli Q water. 100 μLHRP-conjugate was applied to each well, and the plate was covered andincubates at 4° C. for 1 hr. The wash was repeated again for 5 times.100 μL 3,3′,5,5′-Tetramethylbenzidine (TMB) solution was applied to eachwell and the plate was covered and incubated in the dark at roomtemperature for 30 minutes. 100 μL STOP-solution was next applied toeach well, and the plate was read at 450 nm wavelength in aspectrophotometer (Labsystems Multiscan Ascent) within 30 min of addingthe STOP-solution to the wells.

Concentration of Aβ in the samples was determined based on a standardcurve generated from standards containing known concentrations ofsynthetic Aβ40.Those skilled in the art will appreciate thatdiethylamine (DEA) and urea extractions will release soluble Aβ, andinsoluble Aβ respectively. Since the ELISA kit is validated and widelyused, it is accepted that as long as the treatment conditions and assayconditions are the same for each compound tested, then the assay shouldyield consistent robust data for the compounds tested and produceminimal discrepancies.

Data Analysis

To determine the concentration of Aβ40 in the samples, the interpolatedvalues of the samples loaded on plates are multiplied by 20 to accountfor the dilutions made when the volumes of DEA, urea and neutralizationsolution were added up. Values are calculated as percentage change inAβ40 compared to vehicle treated animals.

Bioanalysis of Brain and Plasma Samples

TC was determined in plasma and brain homogenate using UltraPerformanceLC® (UPLC®) chromatography followed by tandem-MS (MS/MS) detection.

Apparatus:

Tecan Genesis RSP 200; Biomek NXP, Beckman Coulter; Sigma 4K15centrifuge; Acquity UPLC, Waters; Sciex API4000 TQ, Applied Biosystems;MS software: Analyst version 1.4.1

Chemicals

Acetonitrile, HPLC-grade, Fluka, No. 34967N; Methanol, HPLC-grade,Sigma-Aldrich, Lot 9003S; Formic acid, HPLC-grade, Riedel-de Haën, Lot51660; Purified water, Millipore Synergy UV

Sample Preparation

Brain homogenate was prepared by homogenizing the brain 1:4 (v/v) withwater:2-propanol:DMSO (50:30:20 v/v/v) followed by centrifugation andcollection of the supernatant. Calibration standards and QC samples wereprepared using a Hamilton robot. 150 μL of ISTD in acetonitrile (1 ng/mLISTD) was added to 25 μL of calibration standards, QC samples and testsamples (plasma and brain homogenate) using a Biomek robot. Aftercentrifugation (6200 g, 4° C., 20 min) 100 μL supernatant from eachsample was transferred to a new plate and mixed with 100 μL water with0.1% formic acid using a Biomek robot (method file InVivo transfer).After a quick centrifugation (6200 g, 4° C., 5 min) the samples wereplaced in the auto-sampler.

UPLC-MS/MS Analysis

MS/MS detection was done with an Applied Biosystems Sciex API 4000instrument in positive-ion electrospray ionisation mode. TC and ISTDwere detected at a parent>daughter mass to charge ratio (m/z). Nitrogenwas used for the nebulizer and collision gases. The peak area correlatedlinearly with the plasma and brain concentration of the analytes in therange of 1.00-1000 ng/mL plasma and 5.00-5000 ng/g brain (corrected fordilution). If the plasma/brain sample drug concentration was above 1000ng/mL or 5000 ng/g, the sample was diluted appropriately in blankplasma/blank brain homogenate before analysis.

Chromatographic System

Analytical columns:

Waters Acquity UPLC HSS C18 SB (pH 2-8) 1.8 μm, 2.1x×30 mm.

Mobile phase A: 0.1% aq. formic acid or 0.1% aq. ammonium hydroxide

Mobile phase B: Acetonitrile with 0.1% aq. formic acid or 0.1% aq.ammonium hydroxide.

Weak wash: Methanol

Strong wash: Acetonitrile/Isopropanol/formic acid (50/50/2 v/v/v)

Flow: 0.6 mL/min

Run time: 3 min.

To waste: 0-0.5 min

Temperature: 40° C.

Gradient:

Time (min) % A % B 0 98 2 0.01 98 2 1.5 5 95 2 5 95 2.2 98 2 3 98 2

Compounds 3 and 5 were admistered at doses of 10 mg/kg p.o. and brainand plasma samples were collected at 3 hours post dose and the followingexposures were measured as described above.

TABLE 3 Results for compound 3 Dose Exp Brain/Plasma Aβ40 (mg/kg) (ng/g)ratio reduction (%) Brain Rat 10 511 0.30 24 Plasma Rat 1682 39 BrainRat 30 2284 0.32 38 Plasma Rat 7056 42

TABLE 4 Results for compound 5 Dose Exp Brain/Plasma Aβ40 (mg/kg) (ng/g)ratio reduction (%) Brain Rat 10 187 0.28 5 Plasma Rat 660 40 Brain Rat30 959 0.29 36 Plasma Rat 3348 49

As shown in tables 3 and 4, compounds of the present invention are ableto penetrate the blood brain barrier and show efficacy in the CNS.

MDCK-MDR1 Assay

The permeability of the test compounds was assessed in MDCK-MDR1 cellsthat were cultured to confluency (4-6 days) in a 96 transwell plate.Test compounds were diluted with the transport buffer (HBSS +1% BSA) toa concentration of 0.5 μM and applied to the apical or basolateral sideof the cell monolayer. Permeation of the test compounds from A to Bdirection or B to A direction was determined in triplicate over a60-minute incubation time at 37° C. and 5% CO2 with a relative humidityof 95%. Test compounds were quantified by LC-MS/MS analysis based on thepeaks area ratios of analyte/IS in both the receiver and donor wells ofthe transwell plate.

The apparent permeability coefficient Papp (cm/s) was calculated usingthe equation:

Papp=(dCr/dt)×Vr/(A×C0)

Where dCr/dt is the cumulative concentration of compound in the receiverchamber as a function of time (μM/s); Vr is the solution volume in thereceiver chamber (0.05 mL on the apical side; 0.25 mL on the basolateralside); A is the surface area for the transport, i.e. 0.0804 cm² for thearea of the monolayer; CO is the initial concentration in the donorchamber (μM).

Compounds are classified Pgp substrates when efflux ratio (Papp BA/PappAB) is ≧2.

TABLE 5 BACE1 activity of selected compounds MDCK-MDR1 Compound effluxratio 1 1.16 2 1.75 3 1.22 4 4.01 5 0.99 6 1.36 7 2.43 8 0.92 9 10.99 100.98 11 2.68 12 1.31 13 0.83

As shown in tables 5, the majority of the exemplified compounds of thepresent invention have MDCK-MDR1 efflux ratios below 2 and are thuslikely to be able to cross the blood brain barrier (E Kerns, L Di,Drug-like Properties: Concepts, Structure Design and Methods (2008)Elsevier).

1. A compound of formula I:

wherein: Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,isoxazolyl, and wherein Ar is optionally substituted with one or moresubstituents selected from halogen, CN, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ fluoroalkyl or C₁-C₆ alkoxy; and R¹ is hydrogen,halogen, C₁-C₃ fluoroalkyl or C₁-C₃ alkyl; or a pharmaceuticallyacceptable salt thereof.
 2. The compound or pharmaceutically acceptablesalt thereof according to claim 1, wherein the compound is of formulaIa:


3. The compound or pharmaceutically acceptable salt thereof according toclaim 1, wherein R¹ is F or H.
 4. The compound or pharmaceuticallyacceptable salt thereof according to claim 1, wherein Ar is optionallysubstituted with one or more F, Cl, Br, CN, C₁-C₃ alkyl, C₁-C₃fluoroalkyl or C₁-C₃ alkoxy.
 5. The compound or pharmaceuticallyacceptable salt thereof according to claim 1, wherein Ar is optionallysubstituted pyridyl.
 6. The compound or pharmaceutically acceptable saltthereof according to claim 1, wherein Ar is optionally substitutedpyrimidyl.
 7. The compound or pharmaceutically acceptable salt thereofaccording to claim 1, wherein Ar is optionally substituted pyrazinyl. 8.The compound or pharmaceutically acceptable salt thereof according toclaim 1, wherein Ar is optionally substituted oxazolyl.
 9. The compoundor pharmaceutically acceptable salt thereof according to claim 1,wherein Ar is optionally substituted thiazolyl.
 10. The compound orpharmaceutically acceptable salt thereof according to claim 1, whereinthe compound is selected from the group consisting of:(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-methylthiazole-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrimidine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide,and(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide.11. A pharmaceutical composition comprising the compound orpharmaceutically acceptable salt thereof according to claim 1 and apharmaceutically acceptable carrier. 12-15. (canceled)
 16. Thepharmaceutical composition of claim 11, wherein said compound is offormula Ia:


17. The pharmaceutical composition of claim 11, wherein said compound isselected from the group consisting of:(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-methylthiazole-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrimidine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide,and(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide.18. A method of treating a neurodegenerative or cognitive disorder ordisease, wherein said method compises administering a therapeuticallyeffective amount of the compound, or pharmaceutically acceptable saltthereof, of claim 1 to a patient in need thereof, wherein saidneurodegenerative or cognitive disorder or disease is selected from thegroup consisting of familial Alzheimer's disease, sporadic Alzheimer'sdisease, preclinical Alzheimer's disease, prodromal Alzheimer's disease,mild cognitive impairment, Down's synsdrome and cerebral amyloidangiopathy.
 19. The method of treating the neurodegenerative orcognitive disorder or disease of claim 18, wherein said compound is offormula Ia.:


20. The method of treating the neurodegenerative or cognitive disorderor disease of claim 18, wherein said compound is selected from the groupconsisting of:(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-methylthiazole-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrimidine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide,(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide,and(S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide.